| Registro completo |
| Provedor de dados: |
Ciência Rural
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| País: |
Brazil
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| Título: |
Canine distemper virus detection by different methods of One-Step RT-qPCR
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| Autores: |
Tozato,Claudia de Camargo
Zadra,Vívian Ferreira
Basso,Caroline Rodrigues
Araújo Junior,João Pessoa
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| Data: |
2016-09-01
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| Ano: |
2016
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| Palavras-chave: |
Canine distemper
Molecular diagnosis
One-Step RT-qPCR
Urine
Centrifugation
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| Resumo: |
ABSTRACT: Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.
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| Tipo: |
Info:eu-repo/semantics/article
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| Idioma: |
Inglês
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| Identificador: |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-84782016000901601
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| Editor: |
Universidade Federal de Santa Maria
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| Relação: |
10.1590/0103-8478cr20150932
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| Formato: |
text/html
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| Fonte: |
Ciência Rural v.46 n.9 2016
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| Direitos: |
info:eu-repo/semantics/openAccess
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